The objectives of the project are to gain a better understanding of the the free radical based mechanisms for enzymic catalysis and the role of the adenosyl radical in initiation of the these reactions. Free-radical reactions are of fundamental importance in biology. Although radicals that are generated adventitiously in living system are detrimentral, nature uses free radicals in order to carry out difficult steps in biosynthesis and metabolism. The adenosylcobalamin (AdoCbl)-dependent enzymes represent one famiily of enzymes that carry out free radical mediated reactions. Ethanolamine ammonia-lyase is an AdoCbl-dependent enzyme from bacteria. The enzyme catalyzes the conversion of ethanolamine to acetaldehyde and ammonia, and bacteria which contain the genes for this enzyme are able to grow on ethanolamine, obtained from the breakdown of phospholipids, as their sole source of carbon, nitrogen and energy. The enzyme is a member of a group of AdoCbl-dependent enzymes which catalyze the interchange of positions of a hydrogen atom and a substituent on adjacent carbon atoms. These enzymes use AdoCbl to initiate the radical-mediated rearrangement through homolysis of the cobalt-carbon bond of AdoCbl to give cob(ll)alamin and the reactive 5'-deoxyadenosyl radical. Some of the free radical intermediates in the reaction of substrates reach concentrations such that they can be observed by electron paramagnetic resonance (EPR) spectroscopic methods. These intermediates will be identified through their characteristic nuclear hyperfine splitting patterns in EPR spectra. Substrate analogs which lead to suicide inactivation of the enzyme by free-radicals that stray from the normal reaction pathway will be identified by spectroscopic methods and by identifiction of the reaction products. Factors leading to a greater than trillion fold acceleration in the rate of cobalt--carbon bond cleavage in enzyme-bound AdoCbl will probed by measurement of the epimerization of a chiral 5'-deutero form of AdoCbl using nuclear magnetic resonance methods. The mechanism for the rearrangement steps in the catalytic cycle will be probed by measurement of nitrogen kinetic isotope effects for substrates using isotope ratio mass spectrometry. Genes for the enzyme from different species of bacteria will be cloned and over expressed in E. coli, and the proteins subjected to screens for crystallization.